Vibrio cholerae O1, a cause of epidemic diarrheal disease, normally resides as an indigenous component of riverine, estuarine and marine ecosystems. In these habitats, it associates with the chitinous exoskeletons of zooplankton. The principal objective of this research project is to characterize the interaction of this human pathogen with a chitin surface. Chitin is an insoluble polymer of N-acetylglucosamine; through the secretion of chitinases, V. cholerae can use chitin as a sole source of carbon and nitrogen in nutrient-poor aquatic habitats. This project will explore the hypothesis that chitin utilization by V. cholerae is a four step process: chemotaxis toward and attachment to the chitin surface; horizontal dispersal of attached bacteria across the surface; vertical growth of the attached population as a biofilm community; and detachment from the biofilm surface and resumption of the planktonic mode-of-growth. The work proposed here will explore each of these hypothesized steps through the combined use of genomic, genetic and cell imaging methods. In collaboration with Professor Gill Geesey at Montana State University, reflected differential interference contrast (DIC) and epifluorescence microscopy will be used to image individual cells as they attach to and spread across a synthetic chitin membrane attached to a laminar flow cell experimental system. Scanning confocal laser microscopy (SCLM) will be employed at the Stanford Biofilm Center to capture the temporal and spatial features of biofilm development on the chitin surface. Microarray expression profiling will be used to identify genes, which comprise a hypothesized detachment regulon. Studies undertaken in collaboration with Professor Saul Roseman at Johns Hopkins University will examine the role of a newly identified chitin sensor histidine kinase protein in each of the four chitin utilization steps. Green fluorescent protein (GFP) promoter fusions will be used to disclose when and where individual genes are expressed on the chitin surface and mutants will be sought that are defective in the chitin utilization program. In the last Specific Aim of the proposed work, these results will be examined using experimental systems that more closely resemble conditions in natural aquatic ecosystems including their relevance for the colonization of swimming copepods. If successful, this project should generate new information about the persistence and control of infectious agents in aquatic reservoirs.